Final reconstructions for individual masked classifications/refinements were selected based on the resolution intercepted with the FSC 0.143 from 3D auto-refine. f, Flow chart of focused classification/refinement using the signal subtraction approach from RELION. Purple box indicates the final high-resolution global reconstruction. Blue box indicates one-Hsp70 loading complex. Yellow boxes indicate the selected class to move forward. e, Schematic workflow of the global cryo-EM map reconstruction. The 60 Å low-pass filtered initial model used to reconstruct the 3D model was adopted from the Hsp90 semi-open conformation structure from the Hsp90:Hop cryo-EM structure. d, Initial model generation for the GR-loading complex. Data in ( b-c) are representative data of at least two independent experiments. c, SDS–PAGE stained with Coomassie blue of the fractions treated with 0.02% (w/v) glutaraldehyde cross-linking for 20 min at room temperature, followed by quenching with 20 mM Tris buffer at pH 7.5. Bottom, SDS–PAGE stained with Coomassie blue of the eluted fractions marked in (top). The discrepancy may be a result of multiple species co-eluted. The apparent molecular weight of the eluent estimated by SEC-MALS is ~370 kDa, although the two-Hsp70 client-loading complex is ~440 kDa. ![]() b, Top, elution profile of gel filtration using SEC-MALS to confirm the homogeneity of the GR-loading complex. The Author(s), under exclusive licence to Springer Nature Limited.Ī, Domain organization of the chaperone proteins in the GR-loading complex. Together with the GR-maturation complex structure 9, we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. ![]() The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. However, to our knowledge, a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Chaperoning GR involves a cycle of inactivation by Hsp70 formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex conversion to an active GR-Hsp90-p23 'maturation' complex and subsequent GR release 8. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity 3-7. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp90 2. Maintaining a healthy proteome is fundamental for the survival of all organisms 1.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |